The present invention relates to drugs for relieving drug-induced nephropathy and hepatitis.
The use of drugs for therapeutic and/or diagnostic purposes has increased year by year, and the drugs used have diversified. These drugs can provide us significant benefits but can also cause substantially harmful effects, especially to kidneys, due to their specific functions described below.
Kidneys weigh less than one percent of the total body weight. From a physiological viewpoint, 25 percent of the total cardiac output flows into the kidneys; 150 liters of primitive urine, up to 50 times the total blood plasma, is filtered through glomeruli per day; and final urine is made by reabsorption, secretion, and metabolism through uriniferous tubules variable in structural and functional heterogeneity. Thus, drugs or their metabolites in blood always circulate, and these substances are concentrated and metabolized in kidneys. Consequently, various highly concentrated metabolites, including original drugs, are distributed in kidneys. Kidneys are likely to be frequently and intensively exposed to drugs. Four types of drugs may induce nephropathy: antimicrobial agents, nonsteroidal agents, contrast agents, and antitumor agents.
The liver can also be easily damaged by drugs. Drug-induced hepatopathy is classified by its onset mechanism into toxic hepatopathy caused by direct attack of drugs or their intermediate metabolites to the liver, and allergic hepatopathy caused by allergic response, type IV delayed allergic response in which T cells are involved. Drug-induced hepatitis is caused by, most frequently, antibiotics, followed by drugs for the central nervous system, drugs for circulatory organs, antitumor agents, hormonal agents, diagnostic agents, etc.
Attempts have been made to relieve drug-induced disorders by using xcex3-globulin, cytochrome C, adenine, SH compounds, vitamin B group, etc., but they are not sufficiently effective. It is very important to clinically cure drug-induced disorders (side effects) because of the interruption of the treatment and the importance of patients"" quality of life (Q.O.L.).
An objective of the present invention is to drugs that effectively relieve or suppress disorders induced by various drugs, especially by antitumor agents.
The inventors have focused on the facts that proteins belonging to the midkine (MK) family such as midkine (MK) and pleiotrophin (PTN) are growth and differentiation factors with multiple functions. The functions include 1) elongation of neurite, 2) activation of fibrinolytic system, 3) strong expression in human cancerous areas, and 4) cure of wounds. Numerous studies have been performed on such proteins in order to find novel pharmaceutical effects.
Midkine was discovered as a product of the gene whose expression was induced in the early stage of the differentiation process with retinoic acid in mouse embryonic tumor cells (Kadomatsu, K. et al., Biochem. Biophys. Res. Commun., 151: 1312-1318, 1988). Pleiotrophin was discovered in the brain of a newborn rat as a heparin-binding protein with neurite elongation ability (Rauvala, H., EMBO J., 8: 2933-2941, 1989). Midkine and Pleiotrophin form a novel class of growth and differentiation factors as heparin-binding proteins. They exhibit 45% homology and are collectively called the MK family (Muramatsu, T., Int. J. Dev. Biol., 37: 183-188, 1993; Muramatsu, T., Develop. Growth and Differ. 36(1): 1-8, 1994). Midkine and Pleiotrophin each exhibits a specific expression pattern in development processes, and is expected to be involved in important physiological activation in differentiation.
The inventors found that MK inhibits cell death caused by antitumor agents in vitro and that MK gene relieves disorders induced by an antitumor agent from the results of an experiment in which an antitumor agent was administered to knockout mice in which MK gene was functionally destroyed. The inventors also found that administering MK or PTN to wild mice relieves the disorders caused by antitumor agents, to complete the invention. The present invention encompasses each invention described in the claims.
In this invention, knockout mice provided an opportunity to investigate how MK gene in the living body fights against disorders caused by drugs and to analyze how each knockout mouse responds to the forced administration of MK at the individual level. Details of MK""s function and mechanism are presently not clear. If MK functions as a trigger protein for the functional cascade of cytokines or growth factors, a very small amount of MK is presumably needed, and the use of knockout mice becomes more important. Recently, a cell surface receptor specifically binding to MK with high affinity (molecular weight 250+kDa) has been discovered. Its characteristics imply that autocrine stimulated by MK in tumor cell proliferation could be mediated by the receptor and would activate the JAK/STAT pathway (Edward, A. R. et al., J. Biol. Chem. 273: 3654-3660, 1998). 
To clarify the relationship between MK and ontogenesis, homozygous MK gene-knocked out mice in which parts of exon 2 and exon 3 are damaged as illustrated in FIG. 1 were prepared (Biochemistry 7, Heisei 8: Volume 68, pp. 1239, 4-P-1244). Those knockout mice did not die during the fetal period and weighed significantly less than heterozygous or wild types (Biochemistry 7, Volume 68, pp. 1239, 4-P-1244, 1996).
Antitumor agents were administered to the knockout mice (simply referred as knockout mice) and wild mice. Survival rate, blood urea nitrogen (BUN) level, and creatinine level of each mouse were compared as indices of disorders after the administration to monitor the ability of MK gene in the living body to relieve disorders caused by antitumor agents. BUN and creatinine levels can be used as indices of functional disorders in kidneys because urea is accumulated in blood due to the reduced renal excretory ability.
In this invention, cisplatin was administered to the knockout mice and wild mice, then BUN level and survival rate were compared. The BUN level of the knockout mice were significantly higher than that of the wild mice. The survival rate of the knockout mice also differed significantly from that of wild mice. The death rate of knockout mice increased by the seventh day after the administration. The rate of abnormal BUN levels in knockout mice that had been forcedly administered MK was significantly lower than that in the group that had been administered physiological saline by the third day after the administration. The effectiveness of MK in suppressing renal cell disorders caused by cisplatin was confirmed by conducting an experiment in vitro using human infantile renal cancer cell lines. An experiment using the wild mice revealed that MK relieved acute hepatopathy due to carbon tetrachloride, and that both PTN and MK effectively suppress nephropathy caused by cisplatin.
These results indicate that proteins of this invention belonging to the MK family effectively relieve or suppress drug-induced nephropathy and hepatopathy.
MK protein (simply referred to as MK) used as an effective ingredient of the pharmaceutical composition of the invention is described in the following references (human MK gene, unexamined published Japanese patent application (JP-A) No. Hei 5-91880; sequences of the human MK gene and protein, JP-A No. Hei 6-217778; MK protein, JP-A No. Hei 5-229957; Muramatsu, T., Develop. Growth and Differ. 36(1), 1-8, 1994). PTN protein used as an effective ingredient of the pharmaceutical composition of the invention is described in the following references (Muramatsu, T., Develop. Growth and Differ. 36(1), 1-8, 1994; Kurtz, A. et al., Critical Reviews in Oncogenesis, 6(2):151-177, 1995). FIGS. 11 and 12 show the amino acid sequence of PTN and MK proteins, respectively. The proteins belonging to the MK family and used as effective ingredients of the pharmaceutical composition of this invention include natural proteins derived from humans, mice, or other mammals, or artificial proteins manufactured by chemical synthesis or genetic engineering. Also, the proteins of the invention that belong to the MK family include proteins or polypeptides which do not cause any changes of the above-described biological activities and differ from proteins derived from nature in the number or the sequences of amino acids. Specifically, the present invention includes proteins corresponding to natural proteins in which the amino acid sequence of natural proteins is partially deleted or replaced by other amino acids, or other amino acids or polypeptide of different length are inserted or added. Amino acids to be replaced or inserted are not limited to natural types.
The expression system using E. coli (Studier, F. W. and Moffatt, B. A., J. Mol. Biol. 189: 113-130, 1989; Studier, F. W. et al., Meth. Enzymol. 185: 60-89, 1990) or the expression system using baculovirus (O""Reilly, D. R. et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford University Press, 1992, Ausubel, F. M. et al. eds., Current Protocol in Molecular Biology, Unit 16.11, Wiley Interscience, 1994) can be used to obtain the MK family proteins that are effective ingredients of the invention using genetic engineering techniques. The inventors employed the expression system using methyl alcohol dependent yeast Pichia pastoris to obtain the MK proteins (refer to JP-A No. Hei 9-95454).
The pharmaceutical composition of the invention contains the MK family protein in an amount effective to prevent or treat nephropathy or hepatopathy caused by drugs. The effective ingredients of the invention can be prepared in a desirable dosage form by mixing with usually used pharmaceutically acceptable carriers, vehicles, diluents, preservatives, stabilizers, buffers, etc.
The pharmaceutical composition of the invention can be administered orally or parenterally. Dosage forms for oral administration include tablets, granules, and capsules. Dosage forms for parenteral administration include injection, suppositories, or percutaneous agents, which are administered intravenously, subcutaneously, intramuscularly, or intraperitoneally.
Physiologically active peptides such as MK or PTN are rapidly digested by protease in digestive tracts in general when they are administered orally. To stabilize MK or PTN in vivo, a hybrid MK or hybrid PTN should be prepared by binding it to water-soluble macromolecules (for example, polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP)). Hybrid constructions of IL-6, TNF-xcex1, etc. have been attempted, and the function has been enhanced by selecting the most suitable hybrid condition (Tsutsumi, Y. et al., Br. J. Cancer. 74: 1090-1095; Tsutsumi, Y. et al., Thoromb. Haemostasis, 77: 168-173, 1997; Tsutsumi, Y. et al., J. Control Release, 33: 447-451, 1995).
The MK family proteins that are effective ingredients of the pharmaceutical compositions of the invention vary depending on the dosage of the causative drug, severity of nephropathy or hepatopathy, age, sex, and weight of the patient when used to prevent or treat nephropathy or hepatopathy caused by drugs. The proteins of the invention can be administered once or several times at a dosage of 1 xcexcg/kg to 100 mg/kg of body weight per day.